Mismatch Repair in Gamma-Proteobacteria

伽玛变形菌中的错配修复

• 批准号: 10116421
• 负责人: Richard Fishel
• 金额: $ 32.76万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116421
• 关键词: Adenine; Bacteria; Binding Proteins; Biochemical; Biology; Biophysics; Cells; Closure by clamp; Complement Factor B; Complex; DNA; DNA Single Strand Break; DNA strand break; Defect; Diffusion; Distant; ESKAPE pathogens; Enterobacter; Escherichia coli; Event; Excision; Excision Repair; Gammaproteobacteria; Gene Mutation; Genes; Genetic Recombination; Genome; Goals; Hereditary Nonpolyposis Colorectal Neoplasms; Human; Image; Imaging Device; Imaging Techniques; Imaging technology; In Vitro; Individual; Kinetics; Klebsiella; Lead; Link; Location; Maintenance; Malignant Neoplasms; Mechanics; Methodology; Methylation; Mismatch Repair; Modeling; Molecular; Motion; Multi-Drug Resistance; Mutation; Nucleotides; Organism; Parents; Pathway interactions; Pattern; Polymerase; Process; Property; Proteins; Reaction; Resolution; Role; Single-Stranded DNA; Site; Slide; Testing; Therapeutic; Time; Visualization; biophysical properties; cancer predisposition; clinically relevant; environmental adaptation; exodeoxyribonuclease; gene repair; genetic analysis; helicase; homologous recombination; in vivo; in vivo imaging; innovation; molecular imaging; pathogenic bacteria; real-time images; recruit; single molecule; stoichiometry; visual tracking

MISMATCH REPAIR IN γ-PROTEOBACTERIA PROJECT SUMMARY / ABSTRACT Mismatch repair (MMR) is a highly conserved genome maintenance process that primarily resolves polymerase misincorporation errors. Mutation of the MMR genes dramatically increase spontaneous mutation rates and have been linked to adaptation as well as associated multi-drug resistance in several pathogenic bacteria. MMR mutations in humans are the cause of the common human cancer predisposition Lynch syndrome as well as numerous sporadic cancers. MMR is an excision-resynthesis process that is initiated at a DNA strand break, which may be hundreds to thousands of nucleotides away from the mismatch. The fidelity of MMR depends on establishing the ssDNA break on the error-containing DNA strand. A subset of γ-proteobacteria, including E.coli and the ESKAPE pathogens Klebsiella and Enterobacter, recently evolved DNA adenine methylation (Dam) and MutH to introduce a ssDNA break onto the newly replicated strand containing a misincorporation error. An important distinction between bacteria that utilize the Dam/MutH MMR Pathway and all other organisms is that dam mutations are lethal in combination with mutation of several homologous recombination genes (synthetic lethality). Similarly, mutations of MMR excision components are lethal in combination with replication editing gene mutations. The mechanisms that lead to MMR-dependent synthetic lethality are largely hypothetical. Deterministic models have historically underpinned MMR mechanisms, where definite complexes and progressions are proposed to complete the biochemical events. Using newly developed single molecule- imaging techniques we showed that the initial steps of MMR rely on random DNA diffusion mechanics that are modulated by the two most highly conserved MMR proteins in terrestrial biology, MutS and MutL. This new application will test the hypothesis that the entire multi-component MMR excision process is Stochastic (fully governed by random processes; the complete opposite of Deterministic). Understanding such biochemical randomness should impact future research and therapeutic strategies targeting MMR. We propose to use real-time single molecule imaging in vitro and in vivo to resolve the mechanics of E.coli MMR and its role in synthetic lethality. The Specific Aims are: 1.) quantitative biophysical imaging of individual MMR component activities, 2.) visualization of the complete MMR excision reaction on single mismatched DNA molecules, and 3.) analysis of MMR component interactions in live cells.

γ-蛋白酶的错配修复 项目总结/摘要 错配修复（MMR）是一种高度保守的基因组维护过程，主要解决 聚合酶错误掺入错误。MMR基因的突变显著增加了自发性 突变率，并已被链接到适应以及相关的多药耐药性在几个 致病菌人类的MMR突变是人类常见癌症的原因 易患林奇综合征以及许多散发性癌症。 MMR是一个切除-再合成过程，在DNA链断裂时启动， 到数千个核苷酸的距离MMR的保真度取决于建立 ssDNA在含有错误的DNA链上断裂。γ-变形菌的一个子集，包括大肠杆菌和 ESKAPE病原体克雷伯氏菌和肠杆菌，最近进化的DNA腺嘌呤甲基化（Dam）和 MutH将ssDNA断裂引入到含有错误掺入错误的新复制链上。 利用Dam/MutH MMR途径的细菌和所有其他MMR途径的细菌之间的重要区别是： 生物体的一个重要特征是，大坝突变与几种同源突变结合在一起是致命的。 重组基因（合成致死性）。类似地，MMR切除成分的突变在MMR患者中是致命的。 与复制编辑基因突变相结合。导致MMR依赖的机制 合成致命性在很大程度上是假设。 历史上，确定性模型一直是MMR机制的基础，在MMR机制中， 提出进行以完成生化事件。使用新开发的单分子- 我们的成像技术表明，MMR的初始步骤依赖于随机DNA扩散力学 这是由两个最高度保守的MMR蛋白在陆地生物学，MutS和MutL的调节。 这个新的应用程序将测试的假设，即整个多成分MMR切除过程是 随机（完全由随机过程控制;与确定性完全相反）。理解 这种生物化学随机性应该会影响未来针对MMR的研究和治疗策略。 我们建议在体外和体内使用实时单分子成像来解决 大肠杆菌MMR及其在合成致死中的作用。具体目标是：（1）。定量生物物理成像 （2）个别MMR组成部分的活动。完全MMR切除反应的可视化 单个错配的DNA分子，和3.）活细胞中MMR组分相互作用的分析。